Iron is an essential nutrient but, when present in excess, may turn into a biohazard by catalyzing the generation of aggressive radicals. Bacteria and eukaryotes have developed elegant homeostatic mechanisms to satisfy their metabolic needs for iron and to prevent the toxicity of iron overload. In mammals, the expression of several proteins of iron metabolism is coordinately regulated at the posttranscriptional level by a mechanism which involves binding of iron-regulatory proteins IRP1 and IRP2 to mRNA “iron-responsive elements” (IREs) (reviewed in references 3
, and 29
). In iron-starved cells, IRE-IRP interactions stabilize transferrin receptor (TfR) mRNA and inhibit translation of ferritin (H and L) mRNAs, thereby promoting cellular iron uptake and preventing iron sequestration.
By contrast, IRE-binding activity decreases in iron-replete cells, resulting in opposite homeostatic responses. Under these conditions, the IRE-binding activity of IRP1 is inactivated upon assembly of a cubane iron-sulfur cluster (9
), while IRP2 undergoes degradation by the proteasome (8
). The regulatory function of IRP1 and IRP2 in mammalian iron metabolism has been examined by gene-targeting experiments. IRP1−/−
mice have been reported to lack any obvious phenotype (30
), while IRP2−/−
mice display aberrant iron homeostasis and accumulate iron in the intestinal mucosa and the central nervous system (23
). Moreover, these mice develop a progressive neurodegenerative disorder, suggesting a prominent role of IRP2 in controlling neuronal iron metabolism.
IRP1 and IRP2 share extensive sequence homology and belong to the family of iron-sulfur cluster isomerases (6
). By analogy to mitochondrial aconitase, a well-characterized enzyme of the citric acid cycle and a member of this family, both IRP1 and IRP2 are projected to contain three compact domains, linked to a fourth domain by a flexible hinge region. However, IRP2 contains an insertion of 73 amino acids (aa), which is encoded by a unique exon, within its domain 1. This cysteine- and proline-rich sequence has been implicated in the mechanism for iron sensing by IRP2 and its iron-mediated degradation. In particular, removal of the 73 aa from IRP2 has been reported to yield a stable molecule (16
). Conversely, insertion of this sequence into an equivalent position within domain 1 apparently rendered IRP1 sensitive to iron-dependent degradation (16
Further mapping of the 73-aa domain showed that mutation of the cysteines at positions 168, 174, and 178 to serines abolished the iron-dependent instability of the IRP1-IRP2 hybrid molecule (16
). These data paved the way for the “cysteine oxidation model” to explain the iron-dependent degradation of IRP2 (15
). According to this model, iron mediates the site-specific oxidation of C168, C174, and C178, which tags the protein for ubiquitination and degradation by the proteasome (15
). In support of this model, recent experiments showed that iron can directly bind to the 73-aa domain in vitro and that this interaction yields oxidized cysteine species (19
). It should also be noted that heme has earlier (7
) and more recently (38
) been proposed to serve as a signal for IRP2 degradation.
To better understand the function of IRP2 as an iron sensor in vivo, we generated stably transfected cell lines expressing epitope-tagged wild-type or mutated IRP2. We provide evidence that neither the three cysteines C168, C174, and C178 nor the entire 73 aa domain is critical for the iron-dependent degradation of IRP2. Instead, we demonstrate that the machinery for IRP2 degradation can be saturated when the protein is expressed at high levels, which may help to reconcile previous data. We show that, unexpectedly, antioxidants can also drive IRP2 toward degradation, and we reveal that the iron-mediated degradation of IRP2 may involve the activity of 2-oxoglutarate-dependent oxygenase(s).